Retinoic acid promotes limb induction through effects on body axis extension but is unnecessary for limb patterning

Abstract

Retinoic acid (RA) is thought to be a key signaling molecule involved in limb bud patterning along the proximodistal or anteroposterior axes functioning through induction of Meis2 and Shh, respectively. Here, we utilize Raldh2-/- and Raldh3-/- mouse embryos lacking RA synthesis to demonstrate that RA signaling is not required for limb expression of Shh and Meis2. We demonstrate that RA action is required outside of the limb field in the body axis during forelimb induction but that RA is unnecessary at later stages when hindlimb budding and patterning occur. We provide evidence for a model of trunk mesodermal RA action in which forelimb induction requires RA repression of Fgf8 in the developing trunk similar to how RA controls somitogenesis and heart development. We demonstrate that pectoral fin development in RA-deficient zebrafish embryos can be rescued by an FGF receptor antagonist SU5402. In addition, embryo ChIP assays demonstrate that RA receptors bind the Fgf8 promoter in vivo. Our findings suggest that RA signaling is not required for limb proximodistal or anteroposterior patterning but that RA inhibition of FGF8 signaling during the early stages of body axis extension provides an environment permissive for induction of forelimb buds.

Figure 1

RA is unnecessary for hindlimb budding and patterning. (A-B) RARE-lacZ expression in E10.5 wild-type (WT) and Raldh2^-/- embryos rescued by brief RA treatment (res -/-); mutant forelimb is smaller than hindlimb which has RA activity nearby in mesonephros. (C-D), Transverse sections through the hindlimbs of the embryos shown in panels A and B. (E-F) Raldh2 and Raldh3 mRNA in transverse sections through wild-type hindlimbs demonstrates that Raldh3 co-localizes with mesonephric RA activity. (G-H) RARE-lacZ expression in a rescued Raldh2^-/-;Raldh3^-/- embryo compared to a rescued Raldh2^-/- embryo demonstrates that the hindlimb develops without mesonephric RA. (I-T), E10.5 wild-type and rescued Raldh2^-/-;Raldh3^-/- (R2-/-;R3-/-) embryos hybridized with probes for Fgf8 (I-J), Shh (K-L), Tbx4 (M-N), Pitx1 (O-P); Meis2 (Q-R); and Hoxa11 (S-T); anterior to left, posterior to right. f, forelimb bud; h, hindlimb bud; lpm, lateral plate mesoderm; m, mesonephros; md, mesonephric duct; mm, mesonephric mesenchyme; n, neural tube; s, somite.

Figure 2

Forelimb induction requires RA signaling in the body axis but not limb mesoderm. (A-B) Transverse sections through the forelimbs of the E10.5 embryos shown in Figure 1a-b. RARE-lacZ expression (C-D) and RA-responsive Cdx1 mRNA (E-F) in E9.5 rescued Raldh2^-/- embryos; wild-type and mutant embryos were stained for the same length of time here and in all other studies. (G-H) RARE-lacZ expression in E8.5 (10-somite) rescued Raldh2^-/- embryo; in the mutant no RA activity is detected in somites or eye which normally express Raldh2 but no other RA-synthesizing enzyme at this stage; brackets indicate the beginning of the forelimb field lying parallel to somites 6-10. (I-J) Transverse section through forelimb field of embryos shown in panels G and H showing no RA activity in limb-field lpm of mutant. (K-L) RARβ is not expressed in lateral plate mesoderm of E8.5 (11-somite) rescued Raldh2^-/- embryo. (M-N) Raldh2 expression at 11-13 somite stages. e, endoderm; f, forelimb field; lpm, lateral plate mesoderm; n, neural tube; s, somite.

Figure 3

RA is required for induction but not A-P patterning of forelimb buds. Tbx5 mRNA in Raldh2^-/- embryos that are unrescued (A-B) or rescued with brief RA treatment (C-D); note smaller forelimb field in rescued mutant compared to wild-type. (E-H) Tbx5 and Uncx4.1 mRNA double-staining in rescued Raldh2^-/- embryos; note delay in Tbx5 expression in rescued mutant. (I-J) Hoxb8 mRNA in rescued Raldh2^-/- embryo; note similar anteroposterior boundary of expression in rescued mutant and wild-type. (K-L) Hand2 mRNA in rescued Raldh2^-/- embryo; note small expression domain in rescued mutant. (M-N) Meis2 mRNA and (O-P) Hoxa11 mRNA in E10.5 wild-type and rescued Raldh2^-/-;Raldh3^-/- embryos. f, forelimb field.

Figure 4

Ectopic Fgf8 expression near the forelimb field following loss of RA. (A) Fgf8 mRNA in 13-somite embryos: wild-type (top panels), unrescued Raldh2^-/- (middle panels), and rescued Raldh2^-/- with brief RA treatment (bottom panels); in mutants note abnormal domain of Fgf8 mRNA in the intermediate mesoderm adjacent to the forelimb field marked by double-arrow in whole-mount and arrow in transverse sections. (B) Fgf8 and Uncx4.1 (somite marker) expression in 10-somite Raldh2^-/- embryos following brief RA rescue (res -/-) or extended RA rescue (ext res -/-); note loss of Fgf8 mRNA in intermediate mesoderm following extended RA rescue and higher levels of RARE-lacZ expression showing that RA activity has been stimulated in intermediate mesoderm. Extended RA treatment also results in comparable Tbx5 mRNA domains in the forelimb fields of 12-somite wild-type and Raldh2^-/- embryos. (C) Expression of Spry2 (a marker of FGF signaling) in 7-somite wild-type and unrescued Raldh2^-/- embryos; arrows in mutants point out expansion of FGF signaling into trunk domain where forelimbs develop. (D) Model for RA signaling based on studies presented here as well as previous findings [4, 5] suggesting that RA acts in the body axis to repress Fgf8 during the 1-10 somite stages to provide an environment permissive for forelimb induction; at the 23-28 somite stages RA signaling has retracted anteriorly and is not involved in hindlimb induction. e, endoderm; f, forelimb bud; h, hindlimb bud; im, intermediate mesoderm (mesonephros); lpm, lateral plate mesoderm; n, neural tube; s, somite.